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E Komor

Publications and source records attributed to E Komor.

At least 19 recordsLinked to original sources
PubMed1997

Cloning of the cDNA for glutaredoxin, an abundant sieve-tube exudate protein from Ricinus communis L. and characterisation of the glutathione-dependent thiol-reduction system in sieve tubes.

Sieve-tube exudate protein (STEP) from Ricinus communis L. seedlings consists of a characteristic set of more than 100 different polypeptides, against which a complex antiserum was raised. This antiserum cross-reacted with dominant protein species (molecular weights 10-30 kDa) present in the sieve-tube exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species. For further elucidation of the nature of individuals STEPs in the sieve tubes the anti-STEP serum was used to screen a cDNA expression library constructed from Ricinus cotyledon mRNA. Two clones that differed in the 3' untranslated region encoded a protein of 11 kDa which showed striking homology to bacterial and eucaryotic glutaredoxin sequences. Glutaredoxin activity was confirmed for the recombinant protein after overexpression in Escherichia coli and characterised in detail in sieve-tube exudate. Michaelis Menten constants (Km) for reduced glutathione and cysteine were 2 mM and 50 microns, respectively. Besides L-cysteine, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate. Glutathione, which is the obligate donor of reduced thiols for glutaredoxin, was present in sieve-tube sap in millimolar concentrations (up to 3 mM) with a ratio of total to oxidised glutathione of 3:1. It is suggested that glutaredoxin and glutathione in sieve tubes prevent oxidative damage and may be involved in redox regulation of sieve-tube proteins.

Amino Acid Sequence↗
PubMed1996

Correlation-peak imaging.

Identification and quantitation in conventional 1H spectroscopic imaging in vivo is often hampered by the small chemical-shift range. To improve the spectral resolution of spectroscopic imaging, homonuclear two-dimensional correlation spectroscopy has been combined with phase encoding of the spatial dimensions. From the theoretical description of the coherence-transfer signal in the Fourier-transform domain, a comprehensive acquisition and processing strategy is presented that includes optimization of the width and the position of the acquisition windows, matched filtering of the signal envelope, and graphical presentation of the cross peak of interest. The procedure has been applied to image the spatial distribution of the correlation peaks from specific spin systems in the hypocotyl of castor bean (Ricinus communis) seedlings. Despite the overlap of many resonances, correlation-peak imaging made it possible to observe a number of proton resonances, such as those of sucrose, beta-glucose, glutamine/glutamate, lysine, and arginine.

Ricinus communis↗
PubMed1985

Solubilization and purification of the ATPase from the tonoplast of Hevea.

The tonoplast-bound ATPase of Hevea brasiliensis (caoutchouc tree) was solubilized with dichloromethan and purified 100-fold with two ammonium sulfate precipitation steps and a G-200 gel filtration step. The resulting ATPase activity eluted according to a molecular mass of approximately 200 kDa and chromatographed at an isoelectric pH of 5.3. Subunits of molecular mass 110 kDa, 68 kDa, 24 kDa and 12 kDa appeared after treatment with 1% sodium dodecyl sulfate or spontaneously during storage of the solubilized ATPase. Dodecyl sulfate/polyacrylamide gel electrophoresis yielded four polypeptides of molecular mass 54 kDa, 66 kDa, 23 kDa and 13 kDa. From protein determination by ultraviolet absorption and Coomassie stain it appears that the 54-kDa and the 66-kDa polypeptides exist in multiple copies. No close resemblance to the membrane-bound ATPase of mitochondria, plastids, plasmalemma, chromaffin granules and synaptic vesicles is seen. No antibody cross-reaction to F1 of bacteria is observed. Therefore it is concluded that the vacuolar ATPase represents a novel type of ATPase. Many properties of the tonoplast-bound ATPase such as pH-dependence, substrate specificity, ion-dependence and inhibitor sensitivity did not change when the enzyme had been solubilized and purified. The phosphatase activity was lost during the purification procedure. The stimulation of ATP-hydrolysis in tonoplast vesicles by uncouplers and ionophores was absent in the solubilized ATPase, and also the stimulation by chloride was significantly reduced. Anion channel blockers, such as triphenyltin and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, which are strong inhibitors of membrane-bound ATPase, fully or partly lost their inhibiting effect after solubilization of the ATPase. These results are interpreted to indicate that ionophores do not directly affect the ATPase molecule, whereas chloride might have a small direct effect on the ATPase besides its effect as a permeating anion.

Adenosine Triphosphatases↗
PubMed1985

Sugar specificity and sugar-proton interaction for the hexose-proton-symport system of Chlorella.

The substrate specificity of the glucose-proton symport system was studied to gain information about the spatial relationship between the binding sites for glucose and proton. Charged glucose analogues such as amino sugars or sugar acids were not transported by the uptake system, with the exception of 2-amino-2-deoxy-D-glucose. This glucosamine was taken up in the charged form in uniport mechanism, i.e. without symport of proton. This result was interpreted to mean that the proton-binding site of the symport system is close to the hydroxyl at carbon 2 of glucose. This interpretation was strengthened by the following facts: The steric position of hydroxyl groups at carbons 1, 2 or 3 of glucose were especially important for efficient transport. O-Methylation was not tolerated at carbon 1, but it was tolerated at carbons 3, 4 or 6. The stoichiometric flow of proton and sugar could be disturbed by removal of hydroxyl group at carbon 1 of glucose. The pH-dependence of sugar transport is sugar-specific, e.g. the amino group at carbon 2 of glucose improves transport at higher pH. The configuration at carbon 2 of glucose influences the specificity for the symported ion. It is concluded that the coupled flow of proton and glucose occurs by simultaneous coordinate movement of both in a transmembrane channel.

Biological Transport↗
PubMed1984

Role of the ATPase of sugar-cane vacuoles in energization of the tonoplast.

Vacuoles of sugar-cane suspension cells contained a tonoplast-bound ATPase which was exclusively located on the cytoplasmic side of the vacuole. Vanadate and diethylstilbestrol had little effect on the vacuolar ATPase. ATP was the optimum substrate for the tonoplast ATPase, but there was also evidence for tonoplast-bound GDP-hydrolyzing and GTP-hydrolyzing enzymes which can interfere with the ATPase assay. Other phosphate anhydrides and esters were not hydrolyzed. The addition of MgATP polarized the tonoplast from about 0 mV to an interior-positive value of about +20 mV; MgADP and MgGTP had much less effect; MgGDP and ATP (in the absence of magnesium) had no effect on the membrane potential. The polarization of the tonoplast was insensitive to valinomycin, nigericin, and inhibitors of plasmalemma ATPase, but was strongly reduced by the uncoupler carbonylcyanide m-chlorophenylhydrazone. These data are interpreted as evidence for the action of tonoplast-bound ATPase as a pump which translocates protons into the vacuoles. The activity of the ATPase was highly specific for MgATP2-; the other important ionic states of ATP:ATP4-, HATP3-, MgHATP-, and Mg2ATP neither stimulated nor inhibited. The same was true for Mg2+. Since the protons were not brought to the catalytic site by protonation of the substrate, the tonoplast-ATPase may pick up the proton for translocation from the cytoplasm. The saturation kinetics for MgATP2- hydrolysis were biphasic, the higher affinity ATPase with Km value of 0.7 mM seems to be the physiologically relevant activity.

Adenosine Triphosphatases↗
PubMed1981

The protonmotive potential difference across the vacuo-lysosomal membrane of Hevea brasiliensis (rubber tree) and its modification by a membrane-bound adenosine triphosphatase.

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.

Adenosine Triphosphatases↗
PubMed1981

Mechanism of uptake of L-arginine by sugar-cane cells.

Suspension cells of sugar cane were used as a model system for cells of higher plants to study the mechanism of L-arginine uptake. The uptake system is specific for the L-arginine molecule in the fully ionized state, i.e. delta-guanidino group and alpha-amino group positively charged and carboxyl group negatively charged. This was concluded because the Km value for uptake increased strongly for: (a) L-arginine analogues which lack the charged carboxyl group (L-arginine methyl ester, agmatin); (b) L-arginine analogues, which lack the charged alpha-amino group (L-arginine acid, gamma-guanidinobutyric acid); (c) L-arginine analogues, which lack the charged delta-guanidino group or gamma-guanidinoxy group (L-citrulline, L-canavanine at neutral and alkaline pH-values). The importance of the positive charge of the delta-guanidino group or gamma-guanidinoxy group was further documented by Km values for L-arginine and L-canavanine at different pH values. Only at pH values where the gamma-guanidinoxy group is protonated, was there an effective uptake of L-canavanine and effective competition of L-canavanine with L-arginine. The length of the L-arginine molecule was less important: slightly larger (L-homoarginine) or shorter analogues (L-lysine) were taken up rather well. A spatial rearrangement at the alpha-carbon (D-ariginine) was, however, not tolerated. The uptake of L-arginine proceeds by electrogenic uniport, there is no evidence for symport or antiport of another molecule (though L-canavanine uptake at neutral pH value causes a transient alkalinization of the suspension medium). Charge equilibration is brought about by efflux of protons and potassium ions.

Amino Acids↗
PubMed1981

Glucose induces two amino acid transport systems in Chlorella.

In autotrophically grown Chlorella cells, glucose induces a hexose transport system but, at the same time, the synthesis of two amino acid transport systems is also induced. Thus, the rates of uptake of glycine, L-alanine, L-proline, and L-serine, all of which compete with each other for entry into the cells, increase more than 100-fold when the algae are pretreated with glucose. The rates of L-arginine and L-lysine uptake increase by a factor of 25 to 50. The accumulation of proline and arginine within the cells amounts to 200- and 600-fold, respectively. Glucose does not cause the positive effect on amino acid uptake by serving as metabolic substrate because the nonmetabolizable 6-deoxyglucose also acts as inducer. Cycloheximide prevents the induction. The induced transport system for the four neutral amino acids has a turnover with a half-life of 7 hr, which corresponds closely to the half-life of the hexose transport system. The transport system for the basic amino acids, on the other hand, disappears with a half-life of 25 hr.

Alanine↗
PubMed1979

The effect of intracellular pH on the rate of hexose uptake in Chlorella.

The rate of hexose uptake by Chlorella is reduced by uncouplers such as carbonyl cyanide p-trifluoromethoxyphenyl hydrazone or dinitrophenol even before concentration equilibrium is reached. The addition of uncouplers changes the membrane potential and the intracellular pH. The membrane potential does not influence the initial velocity of net sugar uptake, whereas manipulation of the cell pH by means of dimethyloxazolidinedione or by butyric acid uncovered a dramatic influence of cell pH on the rate of hexose uptake: at pH values of 7.5--6.8 maximal rate of uptake is observed but at more acid pH a strong inhibition takes place with virtually total blockage of uptake at pH 6.1. The decrease of cell pH to 6.1 in the presence of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone could therefore account for the decrease in hexose transport rate. It was shown that the intracellular pH as such determines the rate of uptake and not the pH difference between inside and outside; the transport rate did not correlate with delta pH.

Biological Transport↗
PubMed1979

Greatly decreased susceptibility of nonmetabolizing cells towards detergents.

The addition of different detergents to Chlorella cells that had previously accumulated 6-deoxyglucose causes a rapid release of the hexose analogue into the medium. This effect is independent of the nature of the detergent and is observed only when the cells are in an energized state. Thus, in the presence of the uncoupler p-trifluoromethoxycarbonylcyanide phenylhydrazone or of inhibitors such as N-ethylmaleimide, the cells show a greatly reduced susceptibility towards detergents. Similarly, the detergent-induced loss of accumulated alpha-aminoisobutyric acid from Saccharomyces cerevisiae and of potassium from Escherichia coli is also strongly affected by the energy state of the cells. The differential susceptibility of energized and nonenergized cells was observed at all detergent concentrations tested. Measurements of substrate efflux at different concentrations of Triton indicated that only Triton monomers are responsible for the increase in permeability. The absorption of [14C]Triton X-100 by Chlorella and the binding of detergent to the cells were measured in the presence of metabolic inhibitors. Again, nonenergized cells bound a significantly lower amount of Triton X-100. The amphiphilic antibiotic nystatin produced effects on cell permeability similar to those of detergents, whereas toluene, which is apolar, gave opposite results.

Aminoisobutyric Acids↗
PubMed1976

The determination of the membrane ptoential of Chlorella vulgaris. Evidence for electrogenic sugar transport.

From data on the accumulation of tetraphenylphosphonium within Chlorella vulgaris cells, it can be estimated that these cells possess a membrane potential of --120 to --150 mV (inside negative). Under anaerobic conditions as well as in the presence of uncoupling agents the membrane potential drops to about -60 to -80 mV. Nystatin (50 mug/ml) abolishes it almost completely. Since it took more than 1 h before the tetraphenylphosphonium equilibrium was reached, this method could not be used to measure relatively fast transient changes in membrane potential. However, the rate of influx of tetraphenylphosphonium is also directly dependent on membrane potential and can be followed within minutes. Using this phenomenon as an indicator for membrane potential a brief transient depolarisation was detected after the addition of sugars taken up by Chlorella via the proton cotransport system. The depolarisation was absent from cells not induced for sugar uptake and induced cells did not show it with substances not transported, like mannitol. The maximal depolarisation observed amounted to about 70 mV; after 1 min, however, the membrane potential returned to a value about 25 mV less negative than the one before sugars was added. The results demonstrate that sugar uptake in Chlorella is electrogenic. The delta pH plus membrane potential measured for Chlorella completely cover the energy required to explain the 1600-fold accumulation of 6-deoxyglucose experimentally observed.

Anaerobiosis↗
PubMed1976

Different proton-sugar stoichiometries for the uptake of glucose analogues by Chlorella vulgaris. Evidence for sugar-dependent proton uptake without concomitant sugar uptake by the proton-sugar symport system.

The uptake of hexoses by Chlorella vulgaris is accompanied by the uptake of protons. For 6-deoxyglucose a stoichiometry of one proton taken up per sugar molecule has been measured, whereas for 1-deoxyglucose approximately two protons are taken up per sugar molecule. It was found that in the presence of 1-deoxyglucose a considerable proportion of "carrier" catalyzes the transport of protons without the concomitant transport of sugar. Presumably, the binding of sugar initiates the translocation of the carrier-proton-sugar complex, but whereas 1-deoxyglucose can still dissociate from the complex at the external side of the cytoplasmic membrane, the translocation of the carrier-proton complex continues. This conclusion was reached since (a) the composition of the translocated carrier-proton-sugar complex is the same for both sugar. Its formation is a first order reaction with respect to protons. (b) When 6-deoxyglucose, present inside cells, is exchanged for external sugar, the exchange ratio is two to one when the external sugar is 1-deoxyglucose, two molecules of 6-deoxyglucose are lost for each molecule of 1-deoxyglucose entering. This result indicates that during uptake of 1-deoxyglucose statistically only each second carrier molecule appearing at the internal side of the cytoplasmic membrane is carrying sugar.

Binding Sites↗
PubMed1974

The hexose-proton cotransport system of chlorella. pH-dependent change in Km values and translocation constants of the uptake system.

The proton concentration in the medium affects the maximal velocity of sugar uptake with a K(m) of 0.3 mM (high affinity uptake). By decreasing the proton concentration a decrease in high affinity sugar uptake is observed, in parallel the activity of a low affinity uptake system (K(m) of 50 mM) rises. Both systems add up to 100%. The existence of the carrier in two conformational states (protonated and unprotonated) has been proposed therefore, the protonated form with high affinity to 6-deoxyglucose, the unprotonated form with low affinity. A plot of extrapolated V(max) values at low substrate concentration versus proton concentration results in a K(m) for protons of 0.14 microM, i.e. half-maximal protonation of the carrier is achieved at pH 6.85. The stoichiometry of protons cotransported per 6-deoxyglucose is close to 1 at pH 6.0-6.5. At higher pH values the stoichiometry continuously decreases; at pH 8.0 only one proton is cotransported per four molecules of sugar. Whereas the translocation of the protonated carrier is strictly dependent on sugar this coupling is less strict for the unprotonated form. Therefore at alkaline pH a considerable net efflux of accumulated sugar can occur. The dependence of sugar accumulation on pH has been measured. The decrease in accumulation with higher pH values can quantitatively be explained by the decrease in the amount of protonated carrier. The properties of the unprotonated carrier resemble strikingly the properties of carrier at the inner side of the membrane. The inside pH of Chlorella was measured with the weak acid 5,5-dimethyl-2, 4-oxazolidinedion (DMO). At an outside pH of 6.5 the internal pH was found to be 7.2. To explain the extent of sugar accumulation it has to be assumed that the membrane potential also contributes to active sugar transport in this alga.

Biological Transport↗